Hypothesis

Short-term treatment with a NAD+ precursor (nicotinamide riboside) combined with an AMPK activator (metformin) prior to dasatinib+quercetin (D+Q) senolytic exposure will increase the clearance of senescent cells that are resistant to D+Q alone by lowering the NAD+/NADH ratio, activating SIRT1, and shifting the Bcl‑2 family balance toward pro‑apoptotic BH3‑only proteins.

Mechanistic Rationale

Senescent cells display heterogeneity in cell‑cycle arrest status, which correlates with variable sensitivity to senolytics [8]. Resistant subsets often up‑regulate anti‑apoptotic Bcl‑2 and MCL‑1 to survive pro‑apoptotic stress. Restoring NAD+ levels rebalances the NAD+/NADH ratio, enhancing SIRT1 deacetylase activity [5]. SIRT1 deacetylates FOXO3a, promoting transcription of BH3‑only proteins such as BIM and PUMA, which neutralize Bcl‑2/MCL‑1 and facilitate mitochondrial outer‑membrane permeabilization. Parallel AMPK activation (e.g., via metformin) further inhibits mTORC1, reduces protein synthesis, and increases autophagy, creating a metabolic state that diminishes the survival advantage of senescent cells. This dual metabolic priming should lower the threshold for D+Q‑induced BCL‑XL/BCL‑2 inhibition, converting resistant senescent cells into a D+Q‑sensitive phenotype.

Experimental Design

A double‑blind, placebo‑controlled trial in adults aged 65‑80 with elevated circulating p16INK4a (>2‑fold age‑matched median) will test three arms over 12 weeks:

1. D+Q alone – dasatinib 100 mg + quercetin 1000 mg orally for 3 consecutive days every 4 weeks (standard regimen).
2. NAD+ + AMPK priming – nicotinamide riboside 300 mg daily + metformin 500 mg BID for 2 weeks preceding each D+Q cycle.
3. Placebo – matching placebo on the same schedule. Primary endpoint: change in peripheral blood p16INK4a‑positive CD4+ T‑cell frequency from baseline to week 12 (flow cytometry). Secondary endpoints: plasma SASP cytokines (IL‑6, IL‑8, MMP‑9), physical performance (Short Physical Performance Battery), and safety labs.

Expected Outcomes

If the hypothesis is correct, the priming arm will show a ≥2‑fold greater reduction in p16INK4a‑positive cells compared with D+Q alone (p<0.01), accompanied by a larger decline in SASP factors and improved performance metrics. The placebo arm should exhibit no significant change. These results would demonstrate that metabolic pre‑conditioning overcomes senolytic resistance.

Falsifiability

A null result—no significant difference in senescent cell clearance between the priming and D+Q‑only arms—would falsify the hypothesis, indicating that NAD+/SIRT1 and AMPK pathways do not sufficiently modulate the apoptotic threshold in human senescent cells under these conditions.

Potential Pitfalls and Mitigations

• Drug interactions: Metformin may affect dasatinib pharmacokinetics; we will monitor dasatinib plasma levels in a subset.
• Compliance: Use of pill counts and electronic caps to ensure adherence.
• Heterogeneity of senescence markers: Supplement p16INK4a with a composite SASP score to capture non‑p16‑driven senescence.

By linking NAD+ restoration, AMPK activation, and the intrinsic apoptotic machinery, this study addresses the critical gap of senolytic resistance and proposes a translatable strategy to enhance senolytic efficacy in aging populations.